Nitric oxide signaling is critical to several physiological functions, and dysfunction in the in this signaling cascade is implicated in multiple diseases such as erectile dysfunction, heart disease, neurodegeneration, stroke, hypertension, and gastrointestinal disease. Activation and deactivation of soluble guanylate cyclase (sGC) is of central importance in nitric oxide (NO) signaling. NO regulates sGC at two levels and this is consistent with numerous pharmacological observations of NO signaling that describe tonic and acute roles for NO. The amplitude and duration of these effects of NO in neuronal signaling, cardiac function, vascular tone and vasodilation are vital to the proper function of these systems, but the mechanism for two NO effects has not been thoroughly investigated. A new paradigm for NO signaling through sGC has emerged. Understanding how sGC switches from a low to high activation state is central to this new paradigm. Our specific aims include: (i) Characterization of NO activation of sGC, with emphasis on studies of the physiological relevance of low and high activity states, (ii) Characterization of the allosteric nucleotide and activator binding site(s), and the role of nucleotide in modulating NO activation of the enzyme, and (iii) determining the effect of oxidative damage to sGC and the role of this in human disease. Experimental approaches will include physical biochemical methods such as mass spectrometry and rapid-reaction kinetics, cloning, expression, purification and characterization of wild type and sitedirected mutants of sGC, and experiments in various cellular systems to extend the findings into an in vivo setting. It is a central goal of this proposal to develop an entirely new understanding of the complex relationship between NO and sGC. We seek to develop a complete molecular level view of sGC activation and deactivation by NO and nucleotides (ATP and GTP). The extension of this work into physiological function will provide a rational basis for the understanding and treatment of NO signaling disorders in human disease.